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RNeasy Fibrous Tissue Midi Kit

For purification of up to 1 mg total RNA from fiber-rich tissues
  • Optimized protocols for fibrous tissues
  • High yields of total RNA
  • High-quality RNA for all downstream applications
  • Removal of genomic DNA using DNase I
The RNeasy Fibrous Tissue Midi Kit includes proteinase K for removing abundant protein in fiber-rich tissue samples, and RNeasy spin columns for purifying up to 1 mg of high-quality RNA. Fibrous tissue samples can be conveniently stabilized using RNAlater RNA Stabilization Reagent or Allprotect Tissue Reagent, and efficiently disrupted using a TissueRuptor or TissueLyser system. For smaller samples, the RNeasy Fibrous Tissue Mini Kit (spin-column binding capacity of 100 µg RNA) is also available.
The RNeasy Fibrous Tissue Midi Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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High-quality RNA from fiber-rich tissues.
RNA was isolated from the indicated rat tissues using the RNeasy Fibrous Tissue Mini Kit. RNA quality was determined by [A] formaldehyde agarose gel electrophoresis and [B] scanning with the Agilent 2100 BioAnalyzer.
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RNeasy Fibrous Tissue Midi procedure.

The RNeasy Fibrous Tissue protocol integrates proteinase K and RNase-free DNase digestion steps during RNA isolation to break down proteins and genomic DNA. This ensures higher yields of high-quality RNA from fiber-rich tissues.

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Real-time gene expression analysis in a variety of fibrous tissues.

RNA was isolated from the indicated rat tissues using the RNeasy Fibrous Tissue Mini Kit. Real-time, quantitative RT-PCR amplification was carried out with 100 ng total RNA using the QuantiTect Probe RT-PCR Kit and primers and probe specific for alpha-actin.

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LightCycler analysis of high-quality RNA from heart.
RNA was isolated from mouse heart using the RNeasy Fibrous Tissue Mini Kit. After reverse transcription using the Omniscript RT Kit, real-time, quantitative, PCR was carried out on the LightCycler System using the QuantiTect SYBR® Green PCR Kit and primers specific for alpha-catenin. Amplification curves are shown for the indicated amounts of total RNA. NTC: no template control.
Performance
RNeasy Fibrous Tissue Kits are optimized for use with fiber-rich tissues with RNA yields from 100 mg of representative fibrous tissues of 80–100 µg from heart, 35–130 µg from muscle, and 50–130 µg from skin. The RNeasy Fibrous Tissue procedure ensures easy and efficient isolation of high-quality total RNA (see figure "High-quality RNA from fiber-rich tissues") suitable for any downstream application, including array analysis and real-time RT-PCR (see figures "Real-time gene expression analysis in a variety of fibrous tissues" and "LightCycler analysis of high-quality RNA from heart").
Principle

RNeasy technology simplifies total RNA isolation by combining the stringency of guanidine-isothiocyanate lysis with the speed and purity of silica-membrane purification. RNeasy Fibrous Tissue Kits are optimized for use with fiber-rich tissues, such as skeletal muscle, heart, and aorta, tissues difficult to lyse due to the abundance of contractile proteins, connective tissue, and collagen. The RNeasy Fibrous Tissue protocol integrates proteinase K and RNase-free DNase digestion steps during RNA isolation to break down proteins and genomic DNA for higher yields of high-quality total RNA. Since the RNeasy procedures enrich for mRNA and other RNA species >200 nucleotides, the total RNA yield does not include 5S rRNA, tRNA, and other low-molecular-weight RNAs, which make up 15-20% of total cellular RNA.

Procedure

Samples of fibrous tissue from 20–250 mg are lysed in a guanidine-isothiocyanate buffer. After dilution of the lysate, the sample is treated with proteinase K. Debris is pelleted by centrifugation. Ethanol is then added to the cleared lysate, and RNA is bound to the RNeasy silica membrane. Traces of DNA that may copurify are removed by a DNase treatment on the RNeasy spin column. DNase and any contaminants are washed away, and up to 1 mg high-quality total RNA is eluted in 150–500 µl RNase-free water (see figure "RNeasy Fibrous Tissue Midi procedure").

Applications

RNeasy Fibrous Tissue Kits provide high-quality total RNA from fiber-rich samples that is suitable for any downstream application, including array analysis and real-time RT-PCR.

Features
Specifications
Applications PCR
Elution volume 150–500 µl
Format Spin column
Main sample type Fibre-rich tissue samples
Processing Manual
Purification of total RNA, miRNA, poly A+ mRNA, DNA or protein RNA
Sample amount 20–250 mg
Technology Silica technology
Time per run or per prep 90 minutes
Yield 35–130 µg
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